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Characterization of Chromobacterium violaceum Isolated from Spoiled Vegetables and Antibiogram of Violacein

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Journal of Advanced Laboratory Research in Biology
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e-ISSN 0976-7614
Volume 2, Issue 1, January 2011
Research Article
Characterization of Chromobacterium violaceum Isolated from Spoiled Vegetables and
Antibiogram of Violacein
G. Rajalakshmi1*, A. Sankaravadivoo2 and S. Prabhakaran3
1*
Department of Biotechnology, Hindusthan College of Arts and Science, Coimbatore, India.
2
Department of Botany, Government Arts College, Coimbatore, India.
3
Department of Biotechnology, Hindusthan College of Arts and Science, Coimbatore, India.
Abstract: Chromobacterium violaceum, a gram-negative, facultative anaerobic, non-sporing coccobacillus,
producing a natural antibiotic called violacein was extracted from Chromobacterium violaceum MTCC 2656,
collection of Chandigarh. The present study reports different types of microbial population along with
Chromobacterium violaceum in 36 samples of spoiled vegetables. In the numerous microbial populations, three
different strains (HMCCC 45, HMCCC 46 and HMCCC 47) were isolated and subjected to various morphological
and biochemical tests. The results reveal that the three strains isolated, HMCCC 46 was similar to the standard
except that HMCCC 46 colonies were dark violet in colour. The antibiotic sensitivity test of HMCCC 46 revealed
that it was resistant to vancomycin, ampicillin, linezolid, and susceptible to colistin, oxacillin, gentamicin,
norfloxacin, chloramphenicol, amikacin.
Keywords: Chromobacterium violaceum, Purple Colony, Violacein, Antibiotic Susceptibility.
1.
Introduction
Chromobacterium violaceum (Neisseriaceae) is a
facultative anaerobic, gram-negative bacillus, first
reported from wet rice paste (Boisbaudran, 1882). They
are abundant in different latitudes like tropical and
subtropical regions and is resident of different sources
like spoiled vegetables, soils and freshwater.
Chromobacterium violaceum associated diseases are
unusual because it is an opportunistic bacterium. Hence
it has been implicated in system infections particularly
tropical latitudes (Richard, 1993). The most
distinguished characteristic of Chromobacterium
violaceum is the production of the purple pigment
known as “violacein” (Bromberg and Duran, 2001).
Even though it is an opportunistic pathogenic
bacterium, its notable product violacein has been
introduced as a therapeutic compound for
dermatological purposes (Caldas et al., 1978).
In this juncture, the present study has been
attempted to characterize the violacein from
*Corresponding author:
E-mail: [email protected]
Chromobacterium violaceum in spoiled vegetables and
to assess the antibiotic potential of violacein against the
commercially available antibiotic discs.
2.
Materials and Methods
2.1 Collection of samples
Five samples of spoiled radish and six samples of
spoiled brinjal were collected from the different local
markets of Coimbatore, India. 10 samples of spoiled
radish and 15 samples of brinjal were directly obtained
from the producers in different rural areas around
Coimbatore. All these samples were collected
aseptically in sterile poly-bags kept in the icebox and
transported to the laboratory for analyses.
2.2 Screening of violacein producing bacteria
10 grams of each sample were homogenized with
90ml of 0.85% sterile physiological saline (w/v) in a
homogeniser for 1 min, followed by the homogenized
samples were serially diluted from 10-1 to 10-8. The
Characterization of C. violaceum Isolated from Vegetables and Violacein
diluents were plated onto nutrient agar plates by spread
plate technique and incubated at 40°C for 24 hrs.
Bacterial colonies which produce pigment were
isolated, plated and detected in the Luria Bertani agar
medium. The screened organisms were cultivated in
MRS agar (HiMedia, India) supplemented with 1%
CaCO3. Purple pigment producing bacteria were
screened from the MRS agar plates and the plates were
counted by using plate count agar (HiMedia, India)
incubated aerobically at 40°C for 48-64 hrs. The purity
of the screened colonies was checked by streaking
again and subculturing on fresh agar plates of the
isolation media, followed by microscopic examinations.
Purified strains were preserved in Luria Bertani slant
using 15% glycerol (v/v) at 15°C for further studies.
2.3 Phenotypic Characterization
Cell morphology and motility of isolates were
determined in a phase contrast microscope (Olympus
CH3-BH-PC, Japan) following the method of Harrigan
(1998). The isolates were subjected to the Gramstaining and tested for catalase production. Preliminary
identification and grouping was done on the basis of
morphology of the colonies evaluated in Luria Bertani
agar medium, based on the ability to grow at different
temperatures viz., 30, 40 and 50°C and to grow in
buffer liquid medium with different pH conditions like
4.0, 5.0, 6.0 and 7.0 (Hungria et al., 2001). Further, the
isolated strains were subjected to phenotypic
characterization,
determination
of the sugar
fermentation pattern, other fermentative activity against
sugars (mannose, fructose, sucrose, lactose and
mannitol), urease test, indole test, methyl red and
Voges-Proskauer test, triple sugar iron test, catalase
test, oxidase test and citrate utilization tests
(Cappuccino et al., 2004).
2.4 Production media
The nutritional requirement of C. violaceum is
necessary to increase the violacein production. Rettori
and Duran (1998) has developed the medium
containing 0.5% D-glucose, 0.5% peptone, 0.2% yeast
extract for the production of violacein pigment. The
suspension of the isolated C. violaceum was inoculated
into 500ml of Rettori and Duran liquid medium and
incubated at 40°C for 14 hours at 100 RPM.
2.5 Extraction of violacein
After the incubation, the cells were harvested for
the extraction and purification of violacein pigment.
The purification of violacein was carried out by the
collection of the aliquot of 100ml broth after 35 hrs
incubation, followed by centrifugation at 10,000g for
10 min. The supernatant was eliminated and 30ml of
commercial ethanol (Merck, India) was added to the
pellet to extract the pigment from the cells. Further, it
was again centrifuged at 8000g for 10 min to separate
the cells and the purification was performed by
J. Adv. Lab. Res. Biol.
Rajalakshmi et al
following the procedure of Duran et al., (1994).
Violacein obtained was dissolved in absolute ethanol
and 0.004% of dimethyl sulfoxide (I) (Merck, India)
and stored at 4°C to protect from the light.
2.6 Antibiotic sensitivity test
Phenotypically characterized strains were tested
against various commercially available antibiotics to
determine the antibiotic sensitivity ranges in
comparison with Chromobacterium violaceum MTCC
2656 strain collected from the Microbial Type Culture
Collection (MTCC), Chandigarh. The antibiotic
sensitivity test was accomplished for isolated strains by
following the methodology of Kirby-Bauer’s NCCLS
(National Committee for Clinical Laboratory Standards,
2000) modified disc diffusion technique. The antibiotic
sensitivity test for isolated strains was performed on
Muller-Hinton agar (HiMedia, India) medium
containing 0.8% (w/v) agar by using certain
commercially available antibiotic discs such as
methicillin (15μg), ampicillin (10μg), amikacin (15μg),
chloramphenicol (30μg), colistin (20μg), cotrimoxazole (20μg), furazolidone (30μg), gentamicin
(15μg), linezolid (20μg), oxacillin (15μg), norfloxacin
(20μg) and vancomycin (20μg). The zone of inhibition
was measured by comparison to the standard strain
Chromobacterium violaceum MTCC 2656 after 24
hours of incubation at 40˚C. Based on the sensitivity to
the respective antibiotic discs, the isolates were
characterized.
3.
Result and Discussion
3.1 Screening for pigment production:
In this study, a total of 36 samples of the spoiled
vegetables (brinjal and radish) was analyzed. In these
spoiled vegetables, the population of bacteria, as well as
the C. violaceum, was ranged between 10-5 to 10-7 CFU
g-1, while the brinjal samples collected from the farmers
shows that the population was less than the 10-5 CFU
g-1, In the serially diluted plates, some strains produced
pigment and some produced non-pigmented colonies in
the same plate. The pigmented colonies show excellent
violet pigment producing capability and concurrently it
also shows antagonistic activity against other colonies
on the same plate. Similar kinds of result were already
reported by (Myers et al., (1992) and Sorenson et al.,
(1985). Based on these characters, three different
isolates were screened on different plates and named as
HMCCC 45, HMCCC 46 and HMCCC 47.
3.2 Identification of the isolates
The screened isolates HMCCC 45, HMCCC 46 and
HMCCC 47 shows typical properties of C. violaceum
such as low convex, violet, smooth, gelatinous colony
morphology on the Nutrient Agar medium. The
screened strains show motility in both soft-agar and
hanging drop methods and there was an absence of
19
Characterization of C. violaceum Isolated from Vegetables and Violacein
spore formation. The results obtained from the
morphological and cultural characteristics reveal that
the three isolates are showing same characteristics and
differed only phenotypically (Table 1). Hence, the
isolate HMCCC 46 only was taken for further studies.
Rajalakshmi et al
3.3 Biochemical and Antibiotic-sensitivity profiling
of the isolate
Biochemical test profiles of strain HMCCC 46 are
listed in Table 2 and Plate 1. The biochemical tests
were conducted in accordance with the method adopted
for Chromobacterium violaceum UCP 1489 (Bailey and
Baron, 1994; Koneman et al., 2001).
Table 1. Morphological and cultural characteristics of HMCCC 45, HMCCC 46 and HMCCC 47.
Test
Simple
Gram staining
Nutrient agar
Hanging drop
Soft – agar
Endospore Staining
Growth at
30°C
40°C
50°C
HMCCC 45
HMCCC 46
HMCCC 47
Rod-shaped
Pink colored
Convex, smooth and violet colonies for HMCCC 45 & 47.
The strain HMCCC 46 had dark violet colonies.
Motile
Motile
Absence of Endospore
Viable
Viable
Less feasible
Table 2. Biochemical profile of HMCCC 46 strain.
S.No.
Test
1.
Indole production
2.
Methyl Red
3.
Voges-Proskauer
4.
Citrate Utilization
5.
Triple Sugar Iron Agar
6.
Hydrogen Sulphide
7.
Catalase
8.
Oxidase
9.
Urease
10.
Starch Hydrolysis
11.
Casein hydrolysis
12.
Gelatin Hydrolysis
13.
Carbohydrate Fermentation
(+) Positive, (-) Negative, (+/-) Variable
Reaction
+
+
Acid Butt, Alkaline slant
+
+
+
+/Ferment Glucose (+/-)
Plate 1. Biochemical profile of HMCCC 46 strain.
J. Adv. Lab. Res. Biol.
20
Characterization of C. violaceum Isolated from Vegetables and Violacein
Antibiotic susceptibility test of the isolate was
performed by agar disc diffusion methods. The results
revealed that the isolate was resistant to vancomycin,
ampicillin and linezolid (Table 3). According to
Sorenson et al., (1985); Kaufman et al., (1986); Hassan
et al, (1993), C. violaceum is considered resistant to
penicillin,
amoxicillin,
clavulanate,
ampicillin,
cephalothin, cefamandole and vancomycin. Resistance
prevalence had been always below 60%. In the present
investigation, HMCCC 46 strain was susceptible to
colistin,
oxacillin,
gentamicin,
norfloxacin,
chloramphenicol and amikacin. Intermediary results
were obtained for furazolidone, co-trimoxazole and
methicillin (Table 3 & Fig. 1).
The results of morphological and biochemical
properties of the screened HMCCC 46 strain shows
similar characters of Chromobacterium violaceum
MTCC 2656 (standard strain) obtained from the
Microbial Type Culture Collection (MTCC),
Rajalakshmi et al
Chandigarh. Hence it can be concluded that the strain
HMCCC 46 was Chromobacterium violaceum.
Table 3. Result of the antibiotic sensitivity of HMCCC 46 strain
compared with C. violaceum MTCC 2656 (standard).
Antibiotic
HMCCC 46
MTCC 2656
Ampicillin
S
R
Amikacin
S
S
Colistin
S
S
Co-trimoxazole
I
R
Chloramphenicol
S
S
Furazolidone
I
R
Gentamicin
S
S
Linezolid
R
R
Methicillin
I
I
Oxacillin
S
S
Norfloxacin
S
S
Vancomycin
R
R
R – Resistance; S – Sensitive; I - Intermediate
40
Antibiotic-sensitivity profiling of isolates
35
Zone of inhibition in Diameter (mm)
30
25
20
15
10
5
ol
e
-tr
Co
ra
z
ol
im
ox
az
id
on
e
lid
Fu
Li
ne
zo
in
ik
ac
Am
ph
e
ni
co
l
ili
n
pi
c
lo
ra
m
Ch
Am
n
ici
m
lo
xa
cin
rf
No
lin
G
en
ta
xa
cil
O
tin
lis
Co
yc
in
nc
om
Va
M
et
hi
cil
lin
0
Antibiotic Discs
Fig. 1. Antibiotic-sensitivity profiling of isolates.
4.
Conclusion
In recent years, emphasis has been placed
especially on the chemical nature of the pigments and
their probable roles in the metabolism of the bacterial
cells. Considerable progress has been made in the
solution of these problems. Relatively few workers
have published results of research designed to
J. Adv. Lab. Res. Biol.
determine the effects of the physical and chemical
environment upon the production of pigments by
various species of bacteria. The production of pigment
was measured by a gross visual comparison of the
amount of pigment of a culture grown upon the test
medium with the pigment produced upon a control
nutrient agar. The molecular mechanisms coordinating
the expression of these characteristics in C. violaceum
21
Characterization of C. violaceum Isolated from Vegetables and Violacein
Rajalakshmi et al
remain to be determined. Compared to conventional
methods, this bacterium leads to an about thirteen-fold
yield of pigment.
[9].
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